Substance specific to human PD-1

ABSTRACT

The present invention relates to a substance specific to human PD-1 comprising a part that recognizes human PD-1, a part that recognizes a membrane protein in cell membrane of human PD-1-expressing cells, and linkers. Since the substance specific to human PD-1 selectively can recognize human PD-1 and a membrane protein on cell membrane of human PD-1-expressing cells and can transmit inhibitory signal of human PD-1, it is useful for therapy and/or prevention of diseases caused by immunopathy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 10/543,323 filed Jul. 25,2005, which is a National Stage Application filed under §371 of PCTApplication No. PCT/2004/000549 filed Jan. 22, 2004. The entiredisclosures of the prior applications are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to antibodies specific to human PD-1,bispecific antibodies comprising antibodies against human T cellreceptor complex or human B cell receptor complex, polynucleotidesencoding them, and the usage of the bispecific antibodies.

BACKGROUND OF THE INVENTION

The immune system acquired the mechanism that can respond to variousforeign antigens. The mechanism takes the diversity of an antigenreceptor by recombination of V, (D) and J fragment in T cells and Bcells. Although this mechanism brought about a result that producesautoreactive lymphocytes, these autoreactive lymphocytes are removed bythe negative selection in thymus or bone marrow, and are furthercontrolled by the self-tolerance mechanism of clonal deletion or anergyin periphery.

Although it is thought that an autoimmune disease is developed by thebreakdown of self-tolerance, the researches using various disease murinemodel have been conducted to elucidate the mechanism of pathogenesis.However, the etiology of an autoimmune disease and the molecularmechanism of self-tolerance remained unclear. In such a situation,existence of the mouse which shows the symptoms of an autoimmune diseasecaused by a single gene deficient is very important for study theetiology of an autoimmune disease from a molecular biological viewpoint.CTLA4−/− mouse which causes lethal systemic lymphocytes infiltration(Waterhouse P., et al., Science, 270:985-988, 1995, Tivol E. A., et al.,Immunity, 3:541-547, 1995), SHP-1 deficient mothaten mice (Shulltz L.D., et al., Cell, 73:1445-1454, 1993), lyn−/−mouse which shows thesymptoms of glomerular nephritis (Hibbs M. L., et al., Cell, 83:301-311,1995), and FCRIIB−/−mouse (Bolland S. & Ravetch J. V., Immunity,13:277-285, 2000) are the representation, and the relations of thesemolecules and self-tolerance has been studied.

The PD-1 gene, which belongs to the immunoglobulin super family, encodesa 55 kDa type I transmembrane protein. Both mouse PD-1 and human PD-1consist of 288 amino acids, and have signal peptide at N terminal (20amino acid) and hydrophobic region in the middle part, which is atransmembrane region (The EMBO J., 11(11):3887-3895, 1992); Japanesepatent Publication No. 5-336973; EMBL/GenBank/DDJB Acc. No. X67914,Genomics, 23:704, 1994; Japanese patent Publication No. 7-291996 (U.S.Pat. No. 5,629,204).

In thymus, PD-1 is expressed at the transition phase between CD4−/CD8−to CD4+/CD8+ stage on thymocytes (Nishimura H., et al., Int. Immunol.,8:773-780 (1996), Nishimura H., et al., J. Exp. Med., 191:891-898(2000)). In periphery, PD-1 is expressed on T cells and B cellsactivated through the antigen receptor (Agata Y., et al., Int. Immunol.,8:765-772 (1996)), and on activated myeloid lineage cells such asmacrophages.

PD-1 has ITIM (Immunoreceptor tyrosine-based inhibitory motif) in itsintracellular region. Therefore, PD-1 is a negative regulator in immuneresponses. Since PD-1 deficient mice developed a lupus-like glomerularnephritis and arthritis (C57BL/6 genetic background) (Nishimura H., etal., Int. Imuunol., 10:1563-1572, 1998, Nishimura H., et al., Immunity,11:141-151, 1999) and dilated cardiomyopathy-like disease (BALB/cgenetic background) (Nishimura H., et al., Science, 291:319-322 (2001)),it became clear that PD-1 serves as a regulator for the development ofautoimmune disease, especially for the maintenance of self tolerance.Further it has been reported that graft rejection is regulated byinhibition of PD-1 signal (Journal of Immunology, 169, 11:6543-6553(2001)).

DISCLOSURE OF THE INVENTION

It is thought that PD-1 is a regulator of various autoimmune diseases,and that it is one of causative genes of autoimmune diseases. Control ofthe PD-1 function can bring about the medical treatment and diagnosis ofsuppression or enhancement of the immune function, infection, transplantrejection, and neoplasm etc. As a result of keen examination, thepresent inventors reached the present invention concerning thesubstances that controls the function of PD-1.

Stimuli on lymphocytes which control immunity are transmitted mainlythrough T cell receptor (TCR) in case of T cells, and B cell receptor(BCR) in case of B cells, and then intracellular phosphorylation playsan important role in the molecular mechanism.

Since it has been elucidated that PD-1 negatively regulates variousimmunocompetent cells such as lymphocytes and myeloid cells etc. andPD-1 has ITIM (Immunoreceptor tyrosine-based inhibitory motif) in itsintracellular region, the present inventors considered that the recruitof de-phosphorylation enzymes (phosphatases) could be involved in themolecular mechanism in the inhibitory signal transduction mediated byPD-1. Therefore, it was hypothesized that making PD-1 colocalize withTCR or BCR can display the PD-1 function.

The present inventors confirmed that the inhibitory signal of PD-1 wastransmitted with the substance which physically cross-links PD-1 to TCRor BCR. First, the present inventors confirmed that the above idea wasright using anti-PD-1 antibodies and anti-CD3 antibodies. CD3 is amembrane protein expressed on T cells and is one component of TCRcomplexes. The divalent antibodies were constructed by bridginganti-PD-1 antibodies and anti-CD3 antibodies. The present invention wascompleted by isolating each cDNA encoding anti-human PD-1 antibody andanti-human TCR antibody and producing bispecific antibody produced byconstructing expression vectors that can express fusions proteinscomprising each antigen-recognition site of both antibodies.

The present invention relates to the followings:

(1) A substance specific to human PD-1 comprising a part that recognizeshuman PD-1, a part that recognizes a membrane protein on cell membraneof human PD-1-expressing cells, and a linker.

(2) The substance specific to human PD-1 according to (1), wherein thepart that recognizes human PD-1 is an antibody against human PD-1 or apartial fragment thereof.

(3) The substance specific to human PD-1 according to (1), wherein thepart that recognizes a membrane protein on cell membrane of humanPD-1-expressing cells is an antibody against the membrane protein or apartial fragment thereof.

(4) The substance specific to human PD-1 according to (1), comprising anantibody against human PD-1 or a partial fragment thereof, an antibodyagainst the membrane protein on cell membrane of human PD-1-expressingcells or a partial fragment thereof, and a linker.(5) The substance specific to human PD-1 according to any one of (1),(3) and (4), wherein the membrane protein is human T cell receptorcomplex or human B cell receptor complex.(6) The substance specific to human PD-1 according to (1) or (4),wherein the linker is a peptide.(7) A bispecific antibody comprising an antibody against human PD-1 or apartial fragment thereof, an antibody against human T cell receptorcomplex or a partial fragment thereof, and a linker.(8) A bispecific antibody comprising an antibody against human PD-1 or apartial fragment thereof, an antibody against human B cell receptorcomplex or a partial fragment thereof, and a linker.(9) The bispecific antibody according to (7) or (8), wherein the linkeris a peptide.(10) A substantially pure polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:2, a homolog thereof, a fragment thereof or ahomolog of the fragment, or a polypeptide comprising the amino acidsequence to which 1-10 amino acid(s) of the polypeptide is/are deleted,substituted, and/or added, wherein the polypeptide composes an antibodyagainst human PD-1.(11) A substantially pure polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:4, a homolog thereof, a fragment thereof or ahomolog of the fragment, or a polypeptide comprising the amino acidsequence to which 1-10 amino acid(s) of the polypeptide is/are deleted,substituted, and/or added, wherein the polypeptide composes an antibodyagainst human PD-1.(12) A polypeptide complex comprising the polypeptides according to (10)and (11).(13) A substantially pure polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:11, a homolog thereof, a fragment thereof or ahomolog of the fragment, or a polypeptide comprising the amino acidsequence to which 1-10 amino acid(s) of the polypeptide is/are deleted,substituted, and/or added.(14) A substantially pure polypeptide comprising the amino acid sequencerepresented by SEQ ID NO:11, a homolog thereof, a fragment thereof or ahomolog of the fragment, or a polypeptide comprising the amino acidsequence to which 1-10 amino acid(s) of the polypeptide is/are deleted,substituted, and/or added, which is the bispecific antibody according toany one of (7) to (9).(15) A polynucleotide encoding the polypeptide according to any one of(10), (11), (13) and (14), a homolog thereof or a complementarypolynucleotide thereof, or a fragment thereof or a homolog of thefragment.(16) A polynucleotide comprising the nucleotide sequence represented bySEQ ID NO:1, SEQ ID NO:3, or SEQ ID NO:9, a homolog thereof or acomplementary polynucleotide thereof, or a fragment thereof or a homologof the fragment.(17) A duplication or expression vector comprising the polynucleotideaccording to (15) or (16).(18) A host cell transformed by the duplication or expression vectoraccording to (17).(19) A manufacturing method of the substance according to any one of (1)to (6), comprising culturing the host cell according to (18) underconditions to express the substance.(20) A manufacturing method of the bispecific antibody according to anyone of (7) to (9), comprising culturing the host cell according to (18)under conditions to express the bispecific antibody.(21) A manufacturing method of the polypeptide according to any one of(10) to (14), comprising culturing the host cell according to (18) underconditions to express the polypeptide.(22) A therapeutic and/or preventive pharmaceutical composition forhuman PD-1 related-diseases, comprising an effective amount of thesubstance according to any one of (1) to (6), the bispecific antibodyaccording to any one of (7) to (9), the polypeptide complex according to(12), or the polypeptide according to (13) or (14).(23) The therapeutic and/or preventive pharmaceutical compositionaccording to (22), wherein the human PD-1 related-diseases are diseasesselected from neurodegenerative disease, autoimmune disease,collagenosis, organ transplantation rejection, tumour, and infectiousdisease.(24) The therapeutic and/or preventive pharmaceutical compositionaccording to (22), wherein the neurodegenerative diseases are diseasesselected from geriopsychosis, Alzheimer disease, Down syndrome,Parkinson's disease, Creutzfeldt-jakob disease, diabetic neuropathy,Parkinson syndrome, Huntington's disease, Machado-Joseph disease,amyotrophic lateral sclerosis, diabetic neuropathy, and CreutzfeldtCreutzfeldt-Jakob disease.(25) The therapeutic and/or preventive pharmaceutical compositionaccording to (22), wherein the autoimmune diseases are diseases selectedfrom glomerular nephritis, arthritis, dilated cardiomyopathy-likedisease, ulceous colitis, Sjogren syndrome, Crohn disease, systemicerythematodes, chronic rheumatoid arthritis, multiple sclerosis,psoriasis, allergic contact dermatitis, polymyosiis, pachyderma,periarteritis nodosa, rheumatic fever, vitiligo vulgaris, insulindependent diabetes mellitus, Behcet disease, Hashimoto disease, Addisondisease, dermatomyositis, myasthenia gravis, Reiter syndrome, Graves'disease, anaemia perniciosa, Goodpasture syndrome, sterility disease,chronic active hepatitis, pemphigus, autoimmune thrombopenic purpura,and autoimmune hemolytic anemia.

The part that recognizes human PD-1 in the present invention has only tobe a substance that recognizes human PD-1, for example, anti-human PD-1antibody or a fragment thereof, human PD-1 itself or a fragment thereof,a ligand for human PD-1 or a fragment thereof (Freeman, G J, et al.,Journal of Experimental Medicine, 19, 7; 1027-1034 (2000)), human PD-L2,and human PD-H3 etc.), and a low molecular weight organic compounds etc.

The antibody for human PD-1 or a fragment thereof may be either ofcomplete polyclonal or monoclonal antibody, humanized antibody, completehuman antibody, and short antibody thereof (for example, F(ab′)₂, Fab′,Fab, Fv) etc. which contains anti-human PD-1 antibody or a fragmentthereof.

Concretely, they are monoclonal anti-human PD-1 antibodies produced byhybridomas (International Trust Number FERM P-19162) that had beendeposited to National Institute of Advanced Industrial Science andTechnology, International Patent Organism Depositary in Central 6, 1-1-1Higashi, Tsukuba, Ibaraki, Japan at Dec. 19, 2002, and had beentransferred to International Deposit at Jun. 5, 2003 (InternationalTrust Number FERM BP-8392), which was named J110. Though F(ab′)₂, Fab′,Fab, and Fv antibody fragments of these antibodies etc. are preferable,they don't limit to them.

F(ab′)₂, Fab′, Fab, and Fv antibody fragments can be obtained byprocessing complete antibodies with protease enzyme, and optionallyreducing it. They can be produced as antibodies, fragments of thereof,or fusion proteins with another proteins and the fragments, by using theexpression vector made by gene rearrangement using the cDNA separatedfrom antibody-producing hybridoma.

The membrane protein on cell membrane of human PD-1-expressing cellsmeans the membrane protein that expresses on cell membrane of the samecell as human PD-1-expressing cells, and includes each the membraneprotein of human PD-1-expressing human first stage culture cells orhuman cell lines. For example, T cell receptor complex or B cellreceptor complex is preferable.

The part that recognizes the membrane protein on cell membrane of humanPD-1-expressing cells may be a substance that recognizes the membraneprotein that expresses on cell membrane of the same cell as humanPD-1-expressing cells, and includes, for example, antibodies against themembrane protein and fragments thereof, the membrane protein itself andfragments thereof, ligands for the membrane protein and fragmentsthereof, and low molecular weight organic compounds that bind to themembrane protein etc.

The antibodies or partial fragments thereof to the membrane protein oncell membrane of human PD-1-expressing cells are antibodies or partialfragments thereof to the membrane protein that expresses on cellmembrane of the same cell as human PD-1-expressing cells, and may beeither of complete polyclonal or monoclonal antibodies, humanizedantibodies, complete human antibodies, and short antibodies thereof (forexample, F(ab′)₂, Fab′, Fab, Fv) etc.

T cell receptor complex is at least a complex comprising T cell receptorconsisting of α subunit and β subunit and CD3 consisting of γsubunit, δsubunit, ε subunit, and ζ subunit.

B cell receptor complex is at least a complex comprising membrane-boundimmunoglobulin, CD79α subunit, and CD790 subunit.

The antibodies or partial fragments thereof to T cell receptor complexmay be either of antibodies or partial fragments thereof that recognizeT cell receptor complex. They include antibodies or partial fragmentsthereof to each subunit of T cell receptor and CD3, which composes Tcell receptor complex and may be either of complete polyclonal ormonoclonal antibodies, humanized antibodies, complete human antibodies,and short antibodies thereof (for example, F(ab′)₂, Fab′, Fab, Fv) etc.For example, the monoclonal antibodies against CD3 can be produced byusing hybridoma that has already been established. Anti-CD3 (α-CD3εmAb(PharMingen, Inc.)) can be bought.

The antibodies or partial fragments thereof to B cell receptor complexmay be either of antibodies or partial fragments thereof that recognizeB cell receptor complex. They include antibodies or partial fragmentsthereof to each subunit of membrane-bound immunoglobulin and CD79, whichcomposes B cell receptor complex, and may be either of completepolyclonal or monoclonal antibodies, humanized antibodies, completehuman antibodies, and short antibodies thereof (for example, F(ab′)₂,Fab′, Fab, Fv) etc. Concretely, commercial anti-BCR antibodies can beused and, for example, anti-IgG (H+ L) polyclonal antibodies can bebought (Zymed Laboratories).

The substance comprising the part that recognizes human PD-1 and thepart that recognizes the membrane protein on cell membrane of humanPD-1-expressing cells means a substance that can simultaneously bind toan extracellular domain of human PD-1 and an extracellular domain of themembrane protein on cell membrane of the same cell. Concretely, theyinclude bispecific antibodies comprising specific antibodies orfragments thereof to human PD-1 and specific antibodies or fragmentsthereof to the membrane protein on cell membrane of humanPD-1-expressing cells.

The bispecific antibodies are unsymmetrical antibodies having twoindependent antigen-recognition sites with two different antigenicspecificities. A well-known chemical method (Nisonoff, A., et al.,Archives of Biochemistry and Biophysics., 90; 460-462 (1961), Brennan,M., et al, Science, 299; 81-83 (1985)) is already well-known as apreparation method of the bispecific antibodies. The bispecificantibodies can be obtained by respectively hydrolyzing two kinds ofantibodies by enzyme and cutting disulfide bonds of the H chains byreducing agents, and then mixing and reoxidizing the different kind ofantibodies. Recently, the preparation method using cross linking agentssuch as glutaraldehyde and carbodiimide has been disclosed (JP02-01556).

Technologies that directly produce the bispecific antibodies by usinggene recombination technologies have been known. For example, theproducing of bispecific antibodies (called single chain diabody) tocarcinoembryonic antigen and E. coli beta-galactosidase has beenreported in Alt, FEBS Letter, 454, 90 (1999). A heavy chain variabledomain (VH) and other light chain variable domain (VL) of the fragmentare connected with linkers to make them combine between two continuousdomains on the same chain. A heavy chain variable domain (VH) and otherlight chain variable domain (VL) of the fragment are connected withlinkers to make them combine between two continuous domains on the samechain. Therefore, VH and VL domain of the fragment are unavoidable tocombine with complementary VL and VH domain of another fragment, as aresult, two antigen binding sites are formed. Though 3-12 amino acidresidues are preferable for the peptide linkers, the sequence is notlimited (Hudson, P J., et al, Journal of Immunology Medicine, 231, 1-2;177-189 (1999)).

As manufacturing of the bispecific antibodies using hybridomas, there isReading, et al's method, that is, a method of producing hybridhybridomas by further fusing two kinds of hybridomas that produce themonoclonal antibodies and then selecting hybrid hybridomas that producetarget bispecific antibodies (U.S. Pat. No. 4,474,893).

The bispecific antibodies comprising specific antibodies or fragmentsthereof to human PD-1 and specific antibodies or fragments thereof tothe membrane protein on cell membrane of human PD-1-expressing cells areantibodies that can simultaneously bind to the extracellular domain ofhuman PD-1 and the extracellular domain of the membrane protein on cellmembrane of the same cell.

The bispecific antibodies can be produced by the following method;

(1) animals are sensitized by human PD-1 or human membrane protein as animmunogen,

(2) splenic cells from the sensitized animals and myeloma cells fromsyngeneic animals are fused,

(3) cells that produce monoclonal antibodies against the sensitizingantigen (human PD-1 or human membrane protein) are screened from theobtained hybridomas,

(4) target antibodies-producing hybridomas are cloned,

(5) the cloned antibodies-producing hybridomas are proliferated,

(6) the produced antibodies are separated and refined,

(7) the bispecific antibodies are produced by cross-linking the obtainedanti-human PD-1 antibodies and anti-human membrane protein antibodieswith linkers, or

(8) they are further digested by pepsin and are separated, and refinedto obtain F(ab′)₂,

(9) each prepared F(ab′)₂ is reduced and is separated and refined,

(10) the bispecific antibodies can be produced by cross-linking eachprepared Fab_(SH) by linkers.

The linkers are not limited and may be anything that can link the partthat recognizes the membrane protein of PD-1-expressing cells to thepart that recognizes human PD-1, keeping an appropriate range. Moreconcretely, they include peptides and amides etc.

The commercial products, for example, phenylenedimaleimide (Aldrich) canbe used as the linkers.

When both or one of substances that respectively recognize the membraneprotein of PD-1-expressing cells and human PD-1 is/are low-molecularweight organic compound(s),

(11) using the antibodies produced by the above technique, thelow-molecules that inhibit a bond between human membrane protein orhuman PD-1 and each antibody are found by measuring with a suitabledetector,

(12) the antibody can be produced by cross-linking the low-molecules,the antibodies, or the Fab with linkers.

It concretely explains each process as follows.

(1) In sensitization, it is preferable that human PD-1 or human membraneprotein is administrated to peritoneal cavity or foot pat of sensitizedanimals. And, the sensitized animals have only to be animals such asmouse and rat etc. that the monoclonal antibody can be obtained ingeneral and are not limited. For example, it is enough that 10-200 μg ofthe antigen are administrated to mouse once.

A spleen of the sensitized animal that antibody titer has risen enoughamong the sensitized animals in previous (1) is removed. Suspension ofthe splenic cells is prepared according to the usual manner. Then, cellfusion of previous (2) is performed by adding polyethylene glycol(preferably PEG4000) to the mixture with the obtained splenic cells andsyngeneic myeloma cells at 37° C. Several kinds such as P3×63Ag8,P3/NS1/1-Ag4-1, and SP-2/0-Ag-14 are known as mouse myeloma cells, andeverything are obtained easily.

It is preferable that myeloma cells are HGPRT (hypoxanthine-guaninephosphoribosyltransferase)⁻ cell lines that can not live in HAT media (amedium containing hypoxanthine, aminopterin, and thymidine), and don'tsecrete antibodies. SP-2/0-Ag-14 are preferably used.

Then, the obtained cell fusion mixtures are sown in 96 microwell plateat low density, which are cultured in HAT medium. By 1-2 weeks, unfusedmyeloma cells, hybridomas of myeloma cells, unfused splenic cells, andhybridomas of splenic cells die out, whereas only hybridomas of spleniccells and myeloma cells proliferate.

By reacting culture supernatant of hybridomas with solid phase antigensand measuring the antibodies that specifically adsorbs the antigens byusing labeled second antibodies, screening of previous (3) judgeswhether hybridomas product the antibodies against human membrane proteinor human PD-1 or not.

The process of (4) is performed by cloning antibody-producing hybridomasaccording to soft agar culture (Monoclonal Antibodies, 372 (1980)). Inthis case, it is also possible to use the limiting dilution.

In the process of (5), to obtain a large amount of antibody moreefficiently, a method of administrating the hybridomas into mouseperitoneal cavity, and separating and refining them from peritonealfluid can be used.

In the process of (6), usual methods such as salting out, ion-exchangechromatography, gel filtration, hydrophobic chromatography, and affinitychromatography etc. are used, and more effectively, affinitychromatography using protein A-sepharose CL-4B (Amersham BiosciencesK.K.) can be used.

Since the bispecific antibodies of the present invention specificallybind, they can be used for purification and concentration such asaffinity chromatography etc. of human PD-1.

In the process of (7), for example, cross linking agents can makesulfo-EMCS (N-(6-maleinimidecaproxy)succinimide sodium salt) bind to SH(mercapto) groups or amide groups of antibodies. First, either ofantibodies is referred to amido coupling reactions with sulfo-EMCS. Theunreacted sulfo-EMCS is separated by gel filtration. SH (mercapto)groups of the other antibody that have been reduced by2-mercaptoethylamine etc. are referred to react with maleimide groups ofsulfo-EMCS that have bound to the first antibody. The one that two kindsof antibodies were mutually cross-linked is separated by gel filtration.

In the process of (8), each antibody that be obtained in process (6) wasdigested by pepsin for 48 hours at 37° C. F(ab′)₂ digested by pepsin arepurified by usual methods such as salting out, ion-exchangechromatography, gel filtration, hydrophobic chromatography, and affinitychromatography etc. and more effectively, gel filtration using cefaclorS-200 (Amersham Biosciences K.K.) can be used.

In the process of (9), F(ab′)₂ are reduced by 2-mercaptoethanol for 30minutes at 30° C. The reduced Fab_(SH) are purified by usual methodssuch as salting out, ion-exchange chromatography, gel filtration,hydrophobic chromatography, and affinity chromatography etc. and moreeffectively, gel filtration using cefaclor S-200 can be used.

In the process of (10), the other antibodies in Fab_(SH) fraction arereferred to bind with linkers. Cross linking agents have only to bindmercapto (SH) groups of Fab_(SH), which is referred to react withphenylenedimaleimide for 30 minutes at room temperature. Then, it isreferred to react with 1.3 times more the other Fab_(SH) for 4 hours atroom temperature. The obtained substance having bivalent specificity arepurified by usual methods such as salting out, ion-exchangechromatography, gel filtration, hydrophobic chromatography, and affinitychromatography etc. and more effectively, gel filtration using cefaclorS-200 can be used.

In the process of (11), the antibodies obtained in process (6) can beused as they are, or as the one that are preferably labeled (forexample, biotin-labeling and FITC-labeling etc.) by the commonprocedure. In ELISA, the antibodies are added to solid phase antigens.Then, when using enzyme-labeled secondary antibody and biotin-labeledantibody, the specific binding between the antibodies and the antigenscan be measured by absorptiometer under the presence of chromophoresubstance, after adding enzyme-labeled streptavidin. Low-molecules thatspecifically recognize PD-1 or the membrane protein can be obtained byusing this assay system.

In the process of (12), when the other is an antibody or Fab, it canbind to an antibody or Fab by introducing suitable functional groups tothe obtained low-molecules. For example, if maleimide groups areintroduced, it can bind to mercapto (SH) groups of the antibody or Fab.If both are low-molecules, molecules including both can be synthesized.

On the other hand, cDNA of each antibody can be separated fromhybridomas that produce the monoclonal antibodies against each antigen.By transforming suitable host cells using expression vector containingDNA that both cDNA or partial fragments thereof have been linked by generecombination methods, the host cells can produce bispecific antibodies.

Concretely, the bispecific antibodies comprising the antibodies againsthuman PD-1 or partial fragments thereof and the antibodies against themembrane protein on cell membrane of human PD-1-expressing cells orpartial fragments thereof can be prepared by the following methods;

(1) Each antibody gene is separated from each hybridoma that anti-humanPD-1 monoclonal antibodies and anti-membrane protein monoclonalantibodies are respectively produced,

(2) Variable region DNA of anti-human PD-1 monoclonal antibody gene andvariable region DNA of anti-membrane protein monoclonal antibody geneare linked by using linker DNA. The expression vectors containing thelinked DNA fragments are introduced to suitable host cells,(3) the cells are cultured under a suitable culture condition, andproduced proteins are separated and refined.

Each process is concretely explained as follows.

The process of (1) comprises a process of separating RNA from hybridomasand a process of separating an antibody gene or cDNA encoding partialpeptide thereof.

The process of separating the total RNA or mRNA from hybridomas can beperformed according to a well-known method (a method described inSambrook, J., et al, Molecular Cloning (1989), Cold Spring HarborLaboratory, or F. M. Ausubel., et al., Current Protocol in MolecularBiology).

By using synthetic DNA primers having partial nucleotide sequences ofthe antibodies of the present invention, cDNA encoding the antibodygenes of the present invention or partial peptide thereof can beamplified by Polymerase Chain Reaction (hereafter, called PCR). Or, byhybridization with probes labeled by using DNA fragments or thesynthetic DNAs encoding a part or whole region of the antibodies of thepresent invention, the cDNA can be selected from cDNA contained insuitable vectors. The hybridization can be performed according to awell-known method. The antibody gene can be amplified by using the totalRNA or mRNA by Reverse Transcriptase Polymerase Chain Reaction(Hereafter, called RT-PCR).

(2) As a method of producing the bispecific antibodies of the presentinvention,

(i) a method of synthesizing peptide and

(ii) a method of producing by gene recombination technologies etc. areenumerated, and the method of the described in (ii) is preferable,industrially.

The expression system (hosts-vector system) to produce peptide by usinggene recombination technologies includes, for example, the expressionsystem of bacillus, yeasts, insect cells, and mammalian cells.

The vector system includes E. coli plasmids (for example, pBR322,pBR325, pUC12, and pUC13), Bacillus subtilis plasmids (for example,pUB110, pTP5, and pC194), yeast plasmids (for example, pSH19 and pSH15),bacteriophages such as lambda phage, animal viruses such as retrovirus,vaccinia virus, and baculovirus, PA1-11, pXT1, pRc/CMV, pRc/RSV, andpcDNAI/Neo etc.

The promoter in the present invention has only to be an appropriatepromoter corresponding to the gene expression host. For example, whenanimal cells are used as hosts, SRα promoter, SV40 promoter, LTRpromoter, CMV promoter, and HSV-TK promoter etc. are enumerated. It ispreferable to use CMV (cytomegalovirus) promoter, SRα promoter etc. Incase of Escherichia coli, trp promoter, lac promoter, recA promoter, λPLpromoter, Ipp promoter, and T7 promoter etc. are preferable. In case ofBacillus bacterium, SPO1 promoter, SPO2 promoter, and penP promoter etc.are preferable. In case of yeasts, PHO5 promoter, PGK promoter, GAPpromoter, and ADH promoter etc. are preferable.

Additionally, the expression vector containing enhancer, splicingsignal, polyA signal, selected marker, and SV40 duplication ori(hereafter, it may be called SV40ori.) etc. can be used, if necessary.The selected marker includes, for example, dihydrofolate reductase(hereafter, it may be called dhfr.) gene (Methotrexate (MTX)resistance), ampicillin resistance gene (hereafter, it may be calledAmpr), and neomycin resistance gene (hereafter, it may be called Neor.G418 resistance) etc. Especially, when dhfr gene is used as a selectedmarker using dhfr gene deficient chinese hamster cells, a target genecan be selected even by using the medium without thymidine. The signalsequence to suitable for the hosts can be added to N-terminal side ofthe protein of the present invention, if necessary. When the hosts areEscherichia, PhoA signal sequence and OmpA signal sequence etc. can beused. When the hosts are bacillus, α-amylase signal sequence andsubtilisin signal sequence etc. can be used. When the hosts arebacillus, MFα signal sequence, SUC2 signal sequence etc. can be used.When the hosts are animal cells, α-insulin signal sequence, α-interferonsignal sequence, and antibody signal sequence etc. can be used,respectively. The transformants can be manufactured using the vectorcontaining DNA encoding the constructed protein of the presentinvention.

By culturing Escherichia transformed with the expression vector insuitable medium, the target peptide is obtained from the body cells.And, if a signal peptide (for example, a signal peptide of pelB) ofbacteria is used, the target peptide is secreted in the periplasm.Further, it can be produced as fusion protein with other peptide. Incase of the expression in mammalian cells, for example, by culturingmammalian cells transformed by suitable expression vectors includingcDNA encoding a target protein in suitable medium, the target peptidesare secreted into medium.

For example, Escherichia, Bacillus bacterium, yeasts, insect cells,insects, and animal cells etc. can be used as host cells. For example,Escherichia coli K12, DH1, JM103, JA221, HB101, C600, JM109, DH5, andDH5α etc. can be used as concrete examples of Escherichia. For example,bacillus Bacillus subtilis MI114 etc. can be used as Bacillus bacterium.For example, Saccharomyces cerevisiae AH22, AH22R-NA87-11A, DKD-5D, or20B-12, Saccharomyces pombe NCYC1913, or NCYC2036, and Pichia pastorisKM71 etc. can be used as yeasts. When virus is AcNPV, for example,Spodoptera frugiperda Cell (SF cell), MG1 cells derived fromTrichoplusia ni midgut, High Five™ cells derived from Trichoplusia niegg, cells derived from Mamestra brassicae, cells derived from Estigmeneacrea etc. can be used as insect cells. When virus is BmNPV, cell linesderived from silkworm (Bombyx mori N cells; BmN cells) etc. can be used.For example, Sf9 cells (ATCC CRL1711) (Vaughn, J. L., In Vivo, 13;213-217 (1977)) and Sf21 cells etc. can be used as Sf cells. Forexample, silkworm larvas etc. can be used as insects. For example,COS-1, COS-7, Vero, chinese hamster cell CHO (hereafter, it'sabbreviated with CHO cells), dhfr-deficient chinese hamster cell CHO(hereafter, it's abbreviated with CHO (dhfr-) cells), mouse L cells,mouse AtT-20, mouse myeloma cells, rat GH3, HEK293 T lymphocytes, andhuman FL cells etc. can be used as animal cells. For example,Escherichia transformation can be performed according to Proc. Natl.Acad. Sci. (USA), 69, 2110 (1972). For example, Bacillus transformationcan be performed according to Molecular & General Genetic, 168, 111(1979). For example, yeasts transformation can be performed according toBecker, D M., et al, Methods in Enzymology, volume 194, p. 182-187(1991), or Proc. Natl. Acad. Sci. (USA), 75, 1929 (1978). Insect cellsor insects transformation can be performed according to Bio/Technology,6; 47-55 (1978). Animal cells transformation can be performed accordingto CELL TECHNOLOGY SUPPLEMENT 8 NEW CELL TECHNOLOGY EXPERIMENTALPROTOCOL, Shujunsha, 263 (1995), or Virology, 52, 456 (1973).

(3) The obtained peptides are purified by usual methods such as saltingout, ion-exchange chromatography, gel filtration, hydrophobicchromatography, and affinity chromatography etc.

In general, a substantially pure polypeptide comprising the amino acidsequence represented by SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:11 is apolypeptide containing the amino acid sequence that is 90% or more, 95,98, or 99% or more homologous to the polypeptide on producing.

A homologue of the substantially pure polypeptide comprising the aminoacid sequence represented by SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:11 isa polypeptide containing the amino acid sequence that is at least 70% ormore, preferably at least 80, 90, or 95% or more homologous to thepolypeptide over a region of at least continuous 20 amino acids,preferably at least 30, 40, 60, or 100 amino acids.

A fragment of the substantially pure polypeptide comprising the aminoacid sequence represented by SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:11 isa polypeptide containing at least 10 amino acids, preferably at least15, 20, 25, 30, 40, 50, or 60 amino acids, and a homologue thereof is apolypeptide containing the amino acid sequence that is at least 70%,preferably at least 80, 90, or 95% or more homologous to the polypeptideover a region of at least continuous 10 amino acids, preferably at leastcontinuous 15, 20, 25, 30, 40, 50, or 60 amino acids.

A substantially pure polypeptide comprising amino acid sequence thatdeleted one or several amino acids from a polypeptide comprising theamino acid sequence represented by SEQ ID NO:2, SEQ ID NO:4 or SEQ IDNO:11 is a polypeptide comprising amino acid sequence that deleted oneor two amino acids or more (preferably 1 to approx. 25, more preferably1 to approx. 10, furthermore, preferably 1 to 5) from amino acidsequence.

A substantially pure polypeptide comprising amino acid sequence that hasbeen substituted by one or several amino acids from a polypeptidecomprising the amino acid sequence represented by SEQ ID NO:2, SEQ IDNO:4 or SEQ ID NO:11 is a polypeptide comprising amino acid sequencethat has been substituted by one or two amino acids or more (preferably1 to approx. 25, more preferably 1 to approx. 10, furthermore,preferably 1 to 5).

A homologue of a polynucleotide comprising DNA the nucleotide sequencerepresented by SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:9 is apolynucleotide that is at least 70%, preferably at least 80, 90, or morepreferably 95% or more homologous to the polynucleotide over a region ofat least continuous 20, preferably at least continuous 30, 40, 60, or100 nucleotide sequence.

A fragment of the polynucleotide comprising DNA the nucleotide sequencerepresented by SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:9 is apolynucleotide containing polynucleotide comprising at least continuous20, preferably at least continuous 30, 40, 50, 60, or 100 nucleotidesequence.

A DNA hybridizing to DNA containing the nucleotide sequence representedby SEQ ID NO:1, SEQ ID NO:3 or SEQ ID NO:9 under stringent conditionincludes, for example, approx. a DNA containing nucleotide sequence thatis approx. 70% or more, preferably approx. 80% or more, more preferablyapprox. 90% or more, most preferably approx. 95% or more homologous tonucleotide sequence respectively represented by SEQ ID NO:1, SEQ IDNO:3, or SEQ ID NO:9.

The hybridization can be performed according to a well-known method or,for example, a method described in J. Sambrook, Molecular Cloning, 2nd,Coldspring Harbor Laboratory. When using a commercial library, it ispossible to perform it according to an attached method described indirections for use. More preferably, it is possible to perform it understringent condition. High-stringent condition is approx. 19-40 mM,preferably approx. 19-20 mM of NaCl concentration, at approx. 50-70° C.,preferably approx. 60-65° C. Approx. 19 mM of sodium concentration atapprox. 65° C. is most preferable, especially.

INDUSTRIAL APPLICABILITY

Application to Medicine:

The substance specific to human PD-1 of the present invention can beused for therapy and/or prevention for the following disease.

The substance specific to human PD-1 of the present invention is usefulfor therapy and/or prevention for neurodegenerative diseases(geriopsychosis, Alzheimer disease, Down syndrome, Parkinson's disease,Creutzfeldt-jakob disease, diabetic neuropathy, Parkinson syndrome,Huntington's disease, Machado-Joseph disease, amyotrophic lateralsclerosis, diabetic neuropathy, and Creutzfeldt Creutzfeldt-Jakobdisease).

The substance specific to human PD-1 of the present invention is usefulfor therapy and/or prevention for diseases that accelerate the immunereaction, for example, autoimmune diseases (glomerular nephritis,arthritis, dilated cardiomyopathy-like disease, ulceous colitis, Sjogrensyndrome, Crohn disease, systemic erythematodes, chronic rheumatoidarthritis, multiple sclerosis, psoriasis, allergic contact dermatitis,polymyosiis, pachyderma, periarteritis nodosa, rheumatic fever, vitiligovulgaris, insulin dependent diabetes mellitus, Behcet disease, Hashimotodisease, Addison disease, dermatomyositis, myasthenia gravis, Reitersyndrome, Graves' disease, anaemia perniciosa, Goodpasture syndrome,sterility disease, chronic active hepatitis, pemphigus, autoimmunethrombopenic purpura, and autoimmune hemolytic anemia).

The substance specific to human PD-1 of the present invention is usefulfor therapy and/or prevention for collagenosis (systemic lupuserythematous, rheumatoid arthritis, systemic scleroderma, diffusescleroderma, dermatomyositis, polymyositis, polymyosiis, Sjogren'ssyndrome, mixed connective tissue disease, polyarteritis nodosa, andrheumatic fever etc.).

The substance specific to human PD-1 of the present invention is usefulfor therapy and/or prevention for organ graft rejection, allergicdisease, and diseases caused by attenuation of immune reaction, whichPD-1 participates, for example, tumour and infectious disease.

When using the substance specific to human PD-1 of the present inventionfor the above purpose, it is usually administered systemically orlocally, and orally or parenterally.

The dosage is different depending on age, body weight, symptom,therapeutic effect, administration route, and duration of the treatmentetc. For oral administration, generally, the dosage range from 0.1 mg to100 mg per an adult is orally administered once to several times perday, or the dosage range from 0.01 mg to 30 mg per an adult isadministered once to several times per day parenterally, suitablyintravenously, and is intravenously administered for 1 to 24 hours perday continuously.

Since the dosage changes depending on various conditions as describedabove, there are cases in which doses lower or greater than the abovedosage may be used.

When a composition of the present invention is administered, it is usedas internal solid medicines and internal liquid medicines for internaluse, and injections, external preparations, suppositoriums etc. forparenteral administration.

The internal solid medicines for oral administration include compressedtablets, pills, capsules, dispersing powders, granules etc. The capsulesinclude hard capsules and soft capsules.

In case of such the solid medicines, one or more active compound(s) maybe pharmaceutically manufactured as itself/themselves or a formulationwith excipients (lactose, mannitol, glucose, microcrystal cellulose, andstarch etc.), binders (hydroxypropylcellulose, polyvinyl pyrrolidone,and magnesium metasilicate aluminate etc.), disintegrators (cellulosecalcium glycolate etc.), lubricants (magnesium stearate etc.),stabilizers, or solubilizers (glutamate and aspartic acid etc.) etc.according to usual methods. Further, they may be optionally coated bycoatings (sucrose, gelatin, hydroxypropylcellulose, andhydroxypropylmethylcellulose phthalate etc.) or coated in the layer oftwo or more. Further, capsules of absorbable materials such as gelatinmay be included.

The liquid compositions for oral administration include pharmaceuticallyacceptable waters, suspensions, emulsions, syrups, and elixirs etc. Asto such liquid medicines, one or more active compound(s) may bedissolved, suspended, or emulsified to generally used diluent (purifiedwater, ethanol or those mixing liquids etc.). Further, those liquidmedicines may contain humectants, suspending agents, emulsifying agents,sweeteners, flavor agents, flavoring agents, preservatives, and buffersetc.

The injections for parenteral administration include solid injectionsthat are dissolved or suspended to solution, suspension, emulsion, ortime of use solvent. The injections are used by dissolving, levigatingand melting one or more activator(s) to the solvent. As the solvent, forexample, water for injection, distilled saline, vegetable oil, propyleneglycol, polyethylene glycol, alcohols such as ethanol etc. and thesecombinations are used. Further, this injection may include stabilizers,solubilizers (glutamate, aspartic acid, and polysorbate 80 (registeredtrademark) etc.), suspending agents, emulsifying agents, soothingagents, buffers, and preservatives etc. These are sterilize in the finalprocess or are manufactured from the aseptic manipulation. Aseptic solidmedicines may be manufactured as a freeze-drying product and may be usedby making to be aseptic or dissolving to aseptic distilled water forinjection or other solvents before use.

Other compositions containing one or more activator(s) for parenteraladministration include liquid for external use, ointment drug, coatingdrug, inhalant, aerosol, suppository and pessary for intrarectaladministration which is prescribed according to a routine procedure.

The sprays may contain a stabilizer such as sodium hydrogen sulfitebesides the diluent generally used, a buffer giving isotonicity, and anisotonic medicine such as, for example, sodium chloride, sodium citrate,or citrates. Production methods of the sprays have been described in,for example, U.S. Pat. No. 2,868,691 specification and U.S. Pat. No.3,095,355 specification in detail.

Since PD-1 relates to the immune reaction, PD-1 can also be used forscreening etc., the substance related to the immune reaction bymeasuring the expression of human PD-1 using the substance specific tohuman PD-1 of the present invention.

The substance specific to human PD-1 of the present invention comprisesof a part that recognizes human PD-1, a part that recognizes themembrane protein on cell membrane of human PD-1-expressing cells, and alinker, and is an excellent substance that specifically recognizes humanPD-1 and the membrane protein, and can transmit human PD-1 signal.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the construction of a bispecific antibody expressionvector.

FIG. 2 shows the reactivity of each bispecific antibody against X63 cellsurface antigen expressed in CD3(−)/PD-1(+) cells and peripheral bloodmononuclear cells (PBMC) surface antigen as CD3(+)/PD-1(−) cells. Inthis figure, filling areas of histogram represent control IgG andopening areas represent distribution of PD-1 or CD3 positive cell.

FIG. 3 shows the effect on proliferative response of activated humanperipheral blood T cells of bispecific antibody. In this figure, “−”represents Control IgG-adding group and “BsAb” represents the bispecificantibody-adding group, the spindle represents measurement absorbance,and ** represents P<0.05 in significance test.

BEST MODE FOR CARRYING OUT THE INVENTION

The following examples explain the present invention more concretely,but don't limit the range of the present invention.

Example 1

To obtain DNA encoding anti-human PD-1 antibody and anti-human CD3antibody respectively, each total RNA was isolated from J110(International Trust Number: FERM BP-8392) hybridoma and CD3 antibodyhybridoma (obtained from ATCC: ATCC Number: CRL-8001). The operation wasperformed by using SV total Isolation System (trade name: purchase fromPromega) according to the manufacturer instructions.

J110 hybridoma cDNA library and CD3 antibody hybridoma cDNA library weregenerated from total RNA (total RNA) by oligo dT prime method by usingReady-To-Go You-Prime First-Strand Beads (trade name: purchase fromAmersham Pharmacia). The operation and procedure were done according tothe manufacturer instructions.

cDNA of variable region of each IgG heavy chain and IgG light chain ofanti-human PD-1 antibody and anti-human CD3 antibody was amplified byPCR reaction using Heavy Primers and Light Primers (the trade name:purchase from Amersham Pharmacia), respectively. PCR was carried out thefollowing steps; as a first step at 95° C. for 2 minutes and as cyclesteps at 95° C. for 30 seconds, at 50° C. for 30 seconds, and at 72° C.for 40 seconds was repeated 30 times and was left at 72° C. for 5minutes as last step.

PCR product was separated by agarose gel electrophoresis. The expectedsize of DNA fragment was collected and was cloned into pGEM-T EasyVector (trade name: purchase from Promega). Then, E. coli DH5 (wastransformed with the plasmid. DNA of each IgG heavy chain (SEQ ID NO:1or 5) and IgG light chain (SEQ ID NO:3 or 7) were sequenced to determinethe consensus sequence.

Example 2

DNAs encoding bispecific antibodies were prepared by being respectivelyconnected to the separated cDNAs in example 1. Fragment 1 was preparedby connecting IgG heavy chain cDNA (SEQ ID NO:1) of anti-human PD-1antibody and IgG light chain cDNA (SEQ ID NO:7) of anti-human CD3antibody by PCR using linker No. 1 (SEQ ID NO:19), linker No. 2 (SEQ IDNO:20), primer No. 1 (SEQ ID NO:14), and primer No. 2 (SEQ ID NO:15)(FIG. 1). Fragment 2 was prepared by connecting IgG heavy chain cDNA(SEQ ID NO:7) of anti-human CD3 antibody and IgG light chain cDNA (SEQID NO:1) of anti-human PD-1 antibody by PCR using linker No. 3 (SEQ IDNO:21), linker No. 4 (SEQ ID NO:22), primer No. 3 (SEQ ID NO:16), andprimer No. 4 (SEQ ID NO:17) (FIG. 1). Fragment 3 was prepared (FIG. 1)by connecting fragment 1 and fragment 2 by PCR using linker No. 5 (SEQID NO:23), linker No. 6 (SEQ ID NO:24), primer No. 5 (SEQ ID NO:18), andprimer No. 4 (SEQ ID NO:22) and then the nucleotide sequence was decided(SEQ ID NO:9).

Each PCR reaction was respectively performed. First PCR was performed byrepeating 20 times the following steps; at 94(C for 30 seconds, at 40(Cfor 30 seconds, and at 72(C for 50 seconds. 2nd PCR was performed byrepeating 30 times the following steps; at 94(C for 30 seconds, at 50(Cfor 30 seconds, and at 72(C for 50 seconds, by using first PCR solutionas a template.

Primer No. 2: (SEQ ID NO: 14) 5′-TTTTTTAAGCTTACAGGTCCAGCTGCAGGAGTCA-3′Primer No. 2: (SEQ ID NO: 15) 5′-TTTTTTGCGGCCGCCCGGTTTATTTCCAACTTTG-3′Primer No. 3: (SEQ ID NO: 16) 5′-TTTTTTAAGCTTACAGGTCCAGCTGCAGCAGTCT-3′Primer No. 4: (SEQ ID NO: 17) 5′-TTTTTTGCGGCCGCCCGTTTGATTTCCAGCTTGG-3′Primer No. 5: (SEQ ID NO: 18) 5′-ATGAACTGGTACCAGCAGAAG-3′ Linker No. 1:(SEQ ID NO: 19) 5′-AGGGACCACGGTCACCGTCTCCTCAGGTGGAGGCGGTTCACAAATTGTTCTCACCCAGTCTCCAG-3′ Linker No. 2: (SEQ ID NO: 20)5′-CTGGAGACTGGGTGAGAACAATTTGTGAACCGCCTCCACCTGAGGAG ACGGTGACCGTGGTCCCT-3′Linker No. 3: (SEQ ID NO: 21)5′-AGGCACCACTCTCACAGTCTCCTCAGGTGGAGGCGGTTCAGACATCC AGATGACCCAGTCTCCAG-3′Linker No. 4: (SEQ ID NO: 22)5′-CTGGAGACTGGGTCATCTGGATGTCTGAACCGCCTCCACCTGAGGAG ACTGTGAGAGTGGTGCCT-3′Linker No. 5: (SEQ ID NO: 23)5′-GGGGACAAAGTTGGAAATAAACCGGGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGGTCCAGCTGCAGCAGTCTGGG G-3′ Linker No. 6: (SEQID NO: 24) 5′-CCCCAGACTGCTGCAGCTGGACCTGCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCCCGGTTTATTTCCAACTTTGTCCC C-3′

DNA encoding the bispecific antibody prepared by the above method wascloned into expression vector pSecTag2/HygroA (trade name: purchase fromInvitrogen). First, each of fragment 1 and fragment 3 was digested byrestriction enzyme HindIII, KpnI, KpnI, and NotI and then was purifiedby agarose electrophoresis. Then, the DNA fragments were ligated in aHindIII and NotI digested pSecTag2/HygroA vector. E. coli DH5 (wastransformed with the plasmid J110-CD3scDb-pSec/hygroA encoding thebispecific antibody was amplified, extracted, and purified (FIG. 1).

Example 3

Bispecific antibody protein was prepared by J110-CD3scDb-pSec/hygroAexpression plasmid. The construct was transiently transfected into humankidney cell line 293T (ATCC Number: CRL-11268) with LipofectAMINE-plus(trade name: purchased from Invitrogen) and cultured for four days. Thesupernatant was sterilized with 0.22 μm PVDF filter, and wasconcentrated using 40% PEG20000 solution. The concentrated supernatantwas purified by HiTrap Chelating HP column (trade name: purchased fromAmershampharmacia).

Example 4

The reactivity of the bispecific antibody (PD-1 and CD3) binding to cellsurface antigens analyzed by FACScan.

1 or 10 μg of the bispecific antibodies were respectively added to humanPD-1-expressed X63 cell lines as PD-1 positive/CD3 negative cells(CD3(−)/PD-1(+) cells) and human peripheral blood mononuclear cells asPD-1 negative/CD3 positive cells (CD3(+)/PD-1(−) cells), and incubatedon ice. Immediately second antibodies were added and incubate on ice for30 min. It was confirmed that the bispecific antibodies reacted to PD-1and CD3.

Example 5

The activity of the bispecific antibody was evaluated as an effect onthe proliferation of activated human peripheral blood T cells.

Concretely, PBMC were isolated from peripheral blood samples of healthydonors by Lymphoprep Tube (trade name: purchased from HYCOMED PHARMA).The operation and procedure were done according to the manufacturerinstructions. Erythricytes were removed from PBMC by lysis with lysisbuffer (0.8% NH₄Cl, 0.1% KCO₃, 1 mM EDTA). And purified T cell passedthrough Nylon Fiber ColumnT (trade name: purchased from Rosh) wereresuspended to medium (RPMI1640 medium including 10% fetal bovineserum).

Purified T cell (2×10⁶/mL/well) were cultured for 60 hours with 1 μg/mlanti-human CD28 antibody (the clone name: CD28.2 and purchased fromPharmingen) in 24 well-plate that has were pre-coated with 5 μg/mlanti-human αβTCR antibody (the clone name: T10B9.1A-31 and purchasedfrom Pharmingen). Activated T cells were rested for 12 hours, andresting T cells (1×10⁶/100 μl/well) were restimulated adding 1 μg/wellof bispecific antibody in 96 well-plate that were pre-coated with 0.1μg/ml anti-αβTCR antibody. 48 hours later, T cell proliferation wasdetermined-by BrdU incorporation using Cell Proliferation ELISA (thetrade name: purchased from Rosh). FIG. 3 shows the result.

The bispecific antibodies have significantly decreased the proliferationof the activated human peripheral blood T cells.

1. A therapeutic method for insulin dependent diabetes mellitus,comprising administering an effective amount of a bispecific antibody toan insulin dependent diabetes mellitus patient, wherein the bispecificantibody comprises the following parts: (a) VH region of anti-human PD-1antibody; (b) VL region of anti-human CD3ε antibody; (c) VH region ofsaid anti-human CD3ε antibody; and (d) VL region of said anti-human PD-1antibody; and wherein said each part is linked by peptide linkers in theabove order, so that said part (a) can bind to human PD-1 together withsaid part (d) and said part (c) can bind to human CD3ε together withsaid part (b).
 2. The preventive and/or therapeutic method according toclaim 1, wherein (a) the VH region of anti-human PD-1 antibody is apolypeptide comprising the amino acid sequence of SEQ ID NO: 2, (b) theVL region of anti-human CD3ε antibody is a polypeptide comprising theamino acid sequence of SEQ ID NO: 8, (c) the VH region of saidanti-human CD3ε antibody is a polypeptide comprising the amino acidsequence of SEQ ID NO: 6 and (d) the VL region of said anti-human PD-1antibody is a polypeptide comprising the amino acid sequence of SEQ IDNO: 4.